Typhoid fever, which is caused by Salmonella typhi, remains an important public health problem for residents in the less developed world, for travelers from industrialized countries who visit endemic areas, and for clinical microbiologists in laboratories which conduct proficiency tests. The currently licensed parenteral killed whole cell typhoid vaccines are protective but cause marked systemic and local adverse reactions at an unacceptably high frequency (Levine, Typhoid fever vaccines, in Plotkin S. A., Mortimer E. A. Jr. (eds): VACCINES. Philadelphia, W. B. Saunders, 1988, pp. 333-361). Alternative vaccines include the recently licensed live oral vaccine strain Ty21a and the experimental parenteral Vi polysaccharide vaccine.
The advantage of an oral vaccine is the delivery of replicating organisms to the mucosal immune system where local responses are maximally stimulated. In addition, attenuated Salmonella typhi are attractive candidates to serve as carrier vaccines to express foreign antigens and deliver them to the human immune system. However, a critical prerequisite for successfully using this approach in immunizing humans is that there must exist highly immunogenic yet safe attenuated strains of S. typhi to deliver the foreign protein and polysaccharide antigens to the immune system.
The current oral vaccine based upon Ty21a has several disadvantages. Ty21a is of relatively low immunogenicity and requires multiple oral doses to immunize. The yield of viable organisms is low when it is fermented and lyophilized in large-scale. In addition, Ty21a has multiple mutations in addition to galE and via, which remain undefined. (Hone et al. (1987), J. Infect. Dis. 156:167-174; Hone et al. (1988), J. Infect. Immun. 56:1326-1333).
Constructs of Ty21a expressing the O antigen of Shigella sonnei (Formal et al. (1981), Infect. Immun. 34:746-750) or the O antigen of Vibrio cholerae 01 serotype Inaba (Forrest et al. (1989), J. Infect. Dis. 159:145-146) have undergone clinical testing in humans. Although two lots of the Ty21a/S. sonnei construct tested in North American volunteers provided significant protection against experimental challenge with pathogenic S. sonnei, there was lot-to-lot variation and other lots were not protective (Black et al. J. Infect. Dis. (1987), 155:1260-1267; Herrington et al. (1990), Vaccine 8:353-357). The Ty21a/Inaba construct elicited serum lnaba vibriocidal antibodies and intestinal SIgA anti-Inaba O antibodies in only a minority of vaccines and at low titer (Tacket et al. (1990), Infect. Immun. 1620-1627). In experimental challenge studies with pathogenic V. cholerae 01, recipients of the construct were not significantly protected overall against diarrhea, but did have milder diarrhea and shed fewer wild-type V. cholerae cells (Tacket et al., Id.).
The main drawbacks to the use of Ty21a as a candidate carrier strain include its limited immunogenicity, a lack of precise information on the molecular basis of its attenuation and practical difficulties in bacterial genetic manipulation of the strain (e.g., in transfomation, electroporation, and recombination frequency). It also exhibits very poor viability after reconstitution of lyophilized cultures.
Applicant has discovered new methods of protecting against virulent infections with vaccines employing transposon-induced avirulent mutants of virulent agents in which the impairment leading to avirulence cannot be repaired by diet or by anything supplied by an animal host. Some of Applicant's initial work, including a method for creating an avirulent microbe by the introduction of deletion mutations in the adenylate cyclase gene (cya) and the cyclic AMP receptor protein gene (crp) of Salmonella typhimurium is described in EPO Pub. No. 315,682 (published 17 May 1989), and PCT Pub. No. WO 88/09669 (published 15 Dec. 1988). Applicant has also provided methods for producing other types of avirulent mutant cells which are desirable as carrier cells for the expression of recombinant antigens. These cells are characterized by a lack of a functioning native gene encoding an enzyme which is essential for cell survival, wherein the enzyme catalyses a step in the biosynthesis of an essential cell wall structural component and the presence of a first recombinant gene encoding an enzyme which is a functional replacement for the native enzyme, wherein the first recombinant gene cannot replace the defective chromosomal gene. In these cells, the first recombinant gene is structurally linked to a second recombinant gene encoding a desired product. Loss of the first recombinant gene causes the cells to lyse. These methods are described in WO 89/03427 (published 20 Apr. 1989). The disclosures of the above-described patent applications, as well as any corresponding national patent applications, are incorporated herein by reference.